Mycobacterium avium subspecies paratuberculosis (a.k.a. M. paratuberculosis) is the etiological agent of Johne's disease, a chronic enteritis of domestic and wild animals, especially ruminants. Johne's disease (JD) has been reported on every continent (1-3), and is considered one of the greatest causes of economic hardship to the ruminant industries (4). More than two thirds of the U.S. dairy herds are infected with JD (5) and this wide distribution of the disease, reduced milk production and premature culling of infected animals together causes severe economic losses estimated to be over $200 million a year for the dairy industry (6,7).
Majority of the M. paratuberculosis infection occurs through fecal-oral route and the mycobacteria are endocytosed by enterocytes and M cells in the Peyer's patches of the ileum (8,9). After subsequent internalization by subepithelial and intraepithelial macrophages, M. paratuberculosis is able to survive and persist within the cells (10) using mechanisms that are not completely understood. Several studies examined gene expression patterns and host defense mechanisms of bovine macrophages from naturally infected cows (11), peripheral blood mononuclear cells (PBMC) (12) or monocytes-derived macrophages (MDMs) (13,14) following infection with M. paratuberculosis. Alternatively, our group characterized the general and specific stress responses of M. paratuberculosis under various in vitro conditions as well as the transcriptomes of M. paratuberculosis in fecal samples from diseased cows (15).
Survival of M. paratuberculosis in environmental samples (16), macrophages (17) and animal models (18,19) is well-documented, however, the genetic basis for this survival remains unknown. Reports employing a large-scale screening of M. paratuberculosis mutants in relevant animal models (20,21) provided some insights into virulence of this organism with the identification of novel virulence factors associated with biofilm formation (22) and epithelial cell invasion (23). Recently, Zhu, et al. analyzed intracellular M. paratuberculosis gene expression patterns in bovine MDMs using SCOTS (selective capture of transcribed sequences), identifying similar patterns of responses to oxidative stress, metabolic activity, and cell survival among M. paratuberculosis with distinct host origins (24). The same group further analyzed the expression profiles of M. paratuberculosis isolated from naturally infected bovine tissues, identifying tissue-specific pathways (25). However, no comprehensive study has been conducted to clarify the relationship between M. paratuberculosis gene expression and specific host microenvironments following macrophage infection.
The current vaccine has a limited use to farmers in some regions (e.g. a few European countries) because of its inability to reduce M. ap shedding in feces of infected animals, the main source for spreading JD. There is only one vaccine (MYCOPAR, Boehringer Ingelheim) approved for limited use in the USA. This vaccine causes significant granuloma formation at the site of inoculation 21, which persists throughout the animal's life, increasing the possibility for tissue condemnation at the slaughterhouse.
Despite the ability of this vaccine to induce cell mediated immunity in animals17, shedding of M. ap from vaccinated animals continues to cause a problem for transmitting the disease to naïve animals (5, 22). In sheep, some animals both shed M. ap and died from multi-bacillary form of JD despite being vaccinated (23). In a long-term study of the effect of killed vaccine on dairy herds to reduce the transmission of the disease, no significant difference in prevalence was found between vaccinated and non-vaccinated herds (22). In another study of commercial JD vaccines, cross reactivity to bovine tuberculosis was prominent, further hampering efforts for controlling tuberculosis in farm animals (24,25). More efforts are needed to better understand the pathogenesis of JD and to plan an effective control strategy.
The present invention starts with a goal to gain insights into how M. paratuberculosis respond to the intracellular microenvironments of macrophages, the primary site for mycobacterial persistence within the host, using targeted mutagenesis and an array of transcriptome analyses. In this study, the inventors took advantage of analytical microscopy to define the phagosome environment of M. paratuberculosis-containing macrophages in association with the expression profile of mycobacterial bacilli using DNA microarrays. The analysis suggested key changes in the metabolic pathways of M. paratuberculosis once the bacteria encounter active macrophages and the activation of various alternative sigma factors (Global Gene Regulators, GGRs) that could help M. paratuberculosis survive the hostile intracellular environment of macrophages. One such alternative GGR, sigH, has been shown to contribute to the resistance encountered during variable environmental stress conditions, such as temperature and oxidative stress in M. tuberculosis (26,27). However, the basis of transcriptional regulation of sigH remains elusive in M. paratuberculosis. 
The inventors therefore sought to define the gene regulatory network under control of GGRs, for example, sigH, sigL, sigE and ECF-1 in M. paratuberculosis. Accordingly, they confirmed a role for these key GGRs activated inside macrophages in defending M. paratuberculosis against thiol-specific oxidative stress and characterized the effect of these GGRs on global transcriptome in M. paratuberculosis. Based on the results, the inventor envisions that the GGR mutants could play an important role in designing effective vaccines against mycobacterial infections.